Programmed cell death (apoptosis) is a process whereby individual cells are terminated during embryonic development and normal growth, to benefit the organism. In general, there are 3 pathways that respond to apoptotic signals and activate protease zymogens (procaspases), which in turn, destroy defined cellular targets. In the mitochondrial pathway, pro-apoptotic signals trigger the release of cytochrome c from the mitochondrial inter-membrane space. Cytochrome c then interacts with the Apoptosis protease activating factor-1 (Apaf-1) in the cytosol to form the apoptosome. Subsequently, the apoptosome recruits procaspase-9 (pc-9) and mediates auto-catalytic conversion to a functional caspase-9. Whilst caspase-9 remains bound to the apoptosome, it can activate procaspase-3 (pc-3) to initiate a proteolytic cascade that leads to cell death. In this proposal, the role of the apoptosome as a cell death organizer will be investigated by determining its 3-dimensional (3D) structure. Electron cryo-microscopy (EM) and single particle image processing will be used to produce 3D maps suitable for molecular modeling. In preliminary 3D studies, we have shown that the apoptosome is assembled from individual Apaf- 1 molecules, to form a central double ring with seven Y-shaped spokes (dimensions: approximately 270 X 70 Angstrom units; mass approximately 1.1 Mda; symmetry yields C7). Based on this structure, we have proposed a model for the activation of Apaf-1 by cytochrome c. In this model, Apaf-1 is auto-inhibited due to interactions between the N-terminal region and two WD40 domains which act as intramolecular chaperones. We suggest that cytochrome c may dock to the WD40 domains within Apaf-1 to release the N-terminal region. This in turn, promotes dATP/ATP binding to the CED4 homology domain and assembly of a functional apoptosome. We will also determine the structures of 3 distinct complexes that contain pc-9, caspase-9/pc-3, and caspase-9 with the mouse X-linked inhibitor of Apoptosis (X-IAP), to provide insights into functional interactions within the apoptosome. In parallel studies, we will attempt to crystallize the apoptosome for X-ray crystallography and utilize a approximately 10 Angstrom units resolution EM map to begin phase refinement by molecular replacement. Together, these studies will provide a detailed understanding of the apoptosome and its interactions with critical cell death components.
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